Over 250,000 people in the United States are currently living with disabilities caused by spinal cord injury (SCI), and approximately 12,000 new injuries are reported each year. Inflammation, including activation of microglia and invasion of macrophages, plays a central role in the secondary injury observed after SCI. After SCI, the NOX family of enzymes is chronically up-regulated and may contribute to microglial/macrophage activation, inflammation and tissue damage through the production of reactive oxygen species (ROS). This expression profile coincides with the invasion of macrophages and activation of microglia into a pro-inflammatory, or M1, phenotype, but not with the M2, or anti-inflammatory, phenotype. Knockout of the NOX2 isoform can reduce ROS production. Further, acute pharmacological inhibition of NOX2 reduces lesion volume following SCI in rats. However, the expression profile of the family of enzymes is currently unclear and the effect of NOX2 inhibition on chronic inflammation, axonal sparing, or motor or autonomic function after SCI is unknown. Therefore, we hypothesize that NOX2 is the primary NOX isoform expressed in M1 microglia/macrophages after spinal cord injury and that inhibition of NOX2 will reduce chronic inflammation and improve recovery, including axonal sprouting/sparing and motor and autonomic function. To test this hypothesis, we propose three specific aims. In aim 1, we will identify the NOX isoform expression profile and correlation with M1 and M2 phenotype after spinal cord injury. In this aim, we will use immunohistochemistry to identify the expression profile of the NOX isoforms and their co-localization with M1 and M2 activation markers. In aim 2, we will demonstrate that NOX2 inhibition induces an M2 phenotype in microglia and macrophages and reduces inflammation in the injured spinal cord. In this aim, we will assess inflammation after moderate SCI at acute, sub-acute and chronic time points using both genetic knockout (gp91PHOX) and pharmacological inhibition approaches. Finally, in aim 3 we will show that delayed administration of a NOX2 inhibitor improves axonal sprouting and sparing and restores motor and autonomic function after spinal cord injury. Utilizing a NOX2 specific inhibitor, we will assess motor and autonomic function, as well as axonal sparing and sprouting. The proposed research study will clarify the role of NOX after SCI and demonstrate the therapeutic applications of NOX inhibition. The data from these studies will provide insight into the role of inflammation, including chronic inflammation, in recovery from SCI. PUBLIC HEALTH RELEVANCE: Over 250,000 people in the United States are currently living with a SCI, and approximately 12,000 new injuries are reported each year. The proposed research study will clarify the role of the NADPH oxidase enzyme (NOX) in the injured spinal cord and demonstrate the therapeutic applications of NOX inhibition. Further, these studies will explore the utility of a delayed treatment approach, capitalizing on the delayed and chronic expression pattern of NOX after SCI, providing the possibility for a more clinically relevant therapeutic approach for SCI treatment.